CysPresso: a classification model utilizing deep learning protein representations to predict recombinant expression of cysteine-dense peptides.

BACKGROUND: Cysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence. RESULTS: We tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning. CONCLUSION: Our novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

Evidence for a Role of Nerve Injury in Painful Intervertebral Disc Degeneration: A Cross-Sectional Proteomic Analysis of Human Cerebrospinal Fluid.

Intervertebral disc degeneration (DD) is a cause of low back pain (LBP) in some individuals. However, although >30% of adults have DD, LBP only develops in a subset of individuals. To gain insight into the mechanisms underlying nonpainful versus painful DD, human cerebrospinal fluid (CSF) was examined using differential expression shotgun proteomic techniques comparing healthy control participants, subjects with nonpainful DD, and patients with painful DD scheduled for spinal fusion surgery. Eighty-eight proteins were detected, 27 of which were differentially expressed. Proteins associated with DD tended to be related to inflammation (eg, cystatin C) regardless of pain status. In contrast, most differentially expressed proteins in DD-associated chronic LBP patients were linked to nerve injury (eg, hemopexin). Cystatin C and hemopexin were selected for further examination using enzyme-linked immunosorbent assay in a larger cohort. While cystatin C correlated with DD severity but not pain or disability, hemopexin correlated with pain intensity, physical disability, and DD severity. This study shows that CSF can be used to study mechanisms underlying painful DD in humans, and suggests that while painful DD is associated with nerve injury, inflammation itself is not sufficient to develop LBP. PERSPECTIVE: CSF was examined for differential protein expression in healthy control participants, pain-free adults with asymptomatic intervertebral DD, and LBP patients with painful intervertebral DD. While DD was related to inflammation regardless of pain status, painful degeneration was associated with markers linked to nerve injury.

Mitochondrial and bioenergetic dysfunction in trauma-induced painful peripheral neuropathy.

BACKGROUND: Mitochondrial dysfunction is observed in various neuropathic pain phenotypes, such as chemotherapy induced neuropathy, diabetic neuropathy, HIV-associated neuropathy, and in Charcot-Marie-Tooth neuropathy. To investigate whether mitochondrial dysfunction is present in trauma-induced painful mononeuropathy, a time-course of mitochondrial function and bioenergetics was characterized in the mouse partial sciatic nerve ligation model. RESULTS: Traumatic nerve injury induces increased metabolic indices of the nerve, resulting in increased oxygen consumption and increased glycolysis. Increased metabolic needs of the nerve are concomitant with bioenergetic and mitochondrial dysfunction. Mitochondrial dysfunction is characterized by reduced ATP synthase activity, reduced electron transport chain activity, and increased futile proton cycling. Bioenergetic dysfunction is characterized by reduced glycolytic reserve, reduced glycolytic capacity, and increased non-glycolytic acidification. CONCLUSION: Traumatic peripheral nerve injury induces persistent mitochondrial and bioenergetic dysfunction which implies that pharmacological agents which seek to normalize mitochondrial and bioenergetic dysfunction could be expected to be beneficial for pain treatment. Increases in both glycolytic acidification and non-glycolytic acidification suggest that pH sensitive drugs which preferentially act on acidic tissue will have the ability to preferential act on injured nerves without affecting healthy tissues.

Peripheral nerve injury induces persistent vascular dysfunction and endoneurial hypoxia, contributing to the genesis of neuropathic pain.

Nerve injury is associated with microvascular disturbance; however, the role of the vascular system has not been well characterized in the context of neuropathic pain. Furthermore, ischemia is thought to play a role in a number of neuropathic pain conditions, and yet the role of hypoxia has also not been characterized in neuropathic pain conditions. In this study, we observed the presence of persistent endoneurial hypoxia in a mouse model of traumatic peripheral nerve injury, causing painful mononeuropathy. We attribute the ongoing hypoxia to microvascular dysfunction, endoneurial fibrosis, and increased metabolic requirements within the injured nerve. Increased lactate levels were observed in injured nerves, as well as increased oxygen consumption and extracellular acidification rates, suggesting that anaerobic glycolysis is required to maintain cellular ATP levels. Hypoxia causes a reduction in levels of the Na(+)/K(+) ATPase ion transporter in both cultured primary dorsal root ganglion neurons and injured peripheral nerve. A reduction of Na(+)/K(+) ATPase ion transporter levels likely contributes to the hyperexcitability of injured nerves. Physiological antagonism of hypoxia with hyperbaric oxygen alleviated mechanical allodynia in nerve-injured animals. These results suggest that hypoxia and the Na(+)/K(+) ATPase ion transporter may be a novel mechanistic target for the treatment of neuropathic pain. In addition, the findings support the possibility of using hypoxia activated pro-drugs to localize treatments for neuropathic pain and nerve injury to injured nerves.

Can modulating inflammatory response be a good strategy to treat neuropathic pain?

Neuronal injury not only results in severe alteration in the function of primary sensory neurons and their central projection pathway, but is also associated with a robust immune response at almost every level of the somatosensory system. Evidence from animal studies suggests undoubtedly that bi-directional signalling between the immune system and the nervous system contribute to the development and maintenance of chronic neuropathic pain. Non-neuronal cells, including peripheral immune cells, CNS/PNS glial cells and endothelial cells play important roles in the neuroimmune interaction and subsequent persistent hypersensitivity. Various cytokines and chemokines have been identified as key signalling molecules in the crosstalk. However, majority evidence showing inflammation in neuropathic pain was generated from animal models at acute phase. Whether and to what extent inflammation or non-neuronal cells are involved at chronic stage of neuropathic pain needs to be further explored, and evidence of inflammation in chronic pain from human studies is still largely awaited. Therapeutic agents targeting inflammation provide an exciting prospect. Yet, considering the heterogeneous conditions presented in neuropathic pain, no matter the etiologies, or the pathophysiology during different stages of the disease; and the complexity of the immune response to the damage on the nervous system, it appears that finely tuned strategies of modulating inflammation are essential to warrant an effective treatment for neuropathic pain. We want to reduce pain; we also want to promote tissue repair and functional recovery.

Blood-nerve barrier dysfunction contributes to the generation of neuropathic pain and allows targeting of injured nerves for pain relief.

The blood-nerve barrier (BNB) is a selectively permeable barrier that creates an immunologically and biochemically privileged space for peripheral axons and supporting cells. The breakdown of the BNB allows access of blood-borne (hematogenous) cells and molecules to the endoneurium to engage in the local inflammatory cascade. This process was examined in a mouse model of trauma-associated neuropathic pain. The impact of nerve injury-triggered opening of the BNB in the development of chronic pain behavior was investigated. Partial ligation of the sciatic nerve led to a long-lasting disruption of the BNB distal to the site of injury. Vascular endothelial growth factor (VEGF) was expressed by resident macrophages after nerve injury. Intraneural injection of VEGF decreased mechanical thresholds while opening the BNB. Serum from nerve-injured or lipopolysaccharide-treated animals elicited mechanical allodynia in naive animals, when allowed to bypass the BNB by intraneural injection. Intraneural injection of fibrinogen, a clotting protein in plasma that was found to deposit in the nerve after nerve injury, also produced a decrease in mechanical thresholds when introduced into naive nerves. These results demonstrate that blood-borne molecules may play a role in the generation of neuropathic pain, suggesting that pain may be driven from infection or injury, at a distance from the nervous system. Furthermore, the breakdown of the BNB in neuropathic conditions was exploited to permit the entry of analgesic molecules that typically cannot pass the BNB, such as ProToxin-II, a BNB-impermeable Nav1.7 inhibitor. Therapeutics utilizing this mechanism could have selective access to injured nerves over healthy tissues.

Salivary biomarkers of HPA axis and autonomic activity in adults with intellectual disability with and without stereotyped and self-injurious behavior disorders.

Salivary levels of biomarkers for the hypothalamic-pituitary-adrenal axis (HPA; cortisol) and sympatho-adreno-medullary system (SAM; α-amylase) were measured in 51 adults (57% male) with neurodevelopmental disorders associated with intellectual disability (i.e., mental retardation) and chronic self-injurious behavior (SIB) and compared with matched controls without SIB. Cortisol levels differed significantly (p < 0.01) between the SIB and control group (SIB > control). Within-group analyses showed significant differences (p < 0.05) in levels of salivary α-amylase between individuals with SIB and those with SIB meeting criteria for stereotyped movement disorder (SMD; SIB + SMD > SIB). Salivary α-amylase was significantly correlated with frequency of stereotypy among the SIB group (r = 0.36, p < 0.05). These preliminary findings warrant further exploration into the role of the SAM system in the pathophysiology of SIB and related repetitive behaviors among individuals with neurodevelopmental disorders associated with intellectual disability.

Statins alleviate experimental nerve injury-induced neuropathic pain.

The statins are a well-established class of drugs that lower plasma cholesterol levels by inhibiting HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase. They are widely used for the treatment of hypercholesterolemia and for the prevention of coronary heart disease. Recent studies suggest that statins have anti-inflammatory effects beyond their lipid-lowering properties. We sought to investigate whether statins could affect neuropathic pain by mediating nerve injury-associated inflammatory responses. The effects of hydrophilic rosuvastatin and lipophilic simvastatin were examined in the mouse partial sciatic nerve ligation model. Systemic daily administration of either statin from days 0 to 14 completely prevented the development of mechanical allodynia and thermal hyperalgesia. When administered from days 8 to 14 after injury, both statins dose-dependently reduced established hypersensitivity. After treatment, the effects of the statins were washed out within 2 to 7 days, depending on dose. Effects of both statins in alleviating mechanical allodynia were further confirmed in a different injury-associated neuropathic pain model, mental nerve chronic constriction, in rats. Both statins were able to abolish interleukin-1ß expression in sciatic nerve triggered by nerve ligation. Additionally, quantitative analysis with Iba-1 and glial fibrillary acid protein immunoreactivity demonstrated that rosuvastatin and simvastatin significantly reduced the spinal microglial and astrocyte activation produced by sciatic nerve injury. The increase of interleukin-1ß mRNA in the ipsilateral side of spinal cords was also reduced by the treatment of either statin. We identified a potential new application of statins in the treatment of neuropathic pain. The pain-alleviating effects of statins are likely attributable to their immunomodulatory effects.

The quaternary lidocaine derivative, QX-314, produces long-lasting local anesthesia in animal models in vivo.

BACKGROUND: QX-314 is a quaternary lidocaine derivative considered to be devoid of clinically useful local anesthetic activity. However, several reports document that extracellular QX-314 application affects action potentials. Hence, the authors tested the hypothesis that QX-314 could produce local anesthesia in animal models in vivo. METHODS: The authors tested QX-314 (10, 30, and 70 mM) in three standard in vivo local anesthetic animal models, using a randomized, blinded experimental design with negative (placebo) and positive (70 mM lidocaine) controls. The guinea pig intradermal wheal assay (n = 29) was used to test for peripheral inhibition of the cutaneous trunci muscle reflex, the mouse tail-flick test (n = 30) was used to test for sensory blockade, and the mouse sciatic nerve blockade model (n = 45) was used to test for motor blockade. RESULTS: In all three animal models, QX-314 concentration-dependently and reversibly produced local anesthesia of long duration, at concentrations equivalent to those clinically relevant for lidocaine. In the guinea pig intradermal wheal assay, QX-314 produced peripheral nociceptive blockade up to 6 times longer than lidocaine (650 +/- 171 vs. 100 +/- 24 min [mean +/- SD]; n = 6 per group; P < 0.0001). In the mouse tail-flick test, QX-314 produced sensory blockade up to 10 times longer than lidocaine (540 +/- 134 vs. 50 +/- 11 min; n = 6 per group; P < 0.0001). Finally, in the mouse sciatic nerve model, QX-314 produced motor blockade up to 12 times longer compared with lidocaine (282 +/- 113 vs. 23 +/- 10 min; n = 9 or 10 per group; P < 0.0001). The onset of QX-314-mediated blockade was consistently slower compared with lidocaine. Animals injected with saline exhibited no local anesthetic effects in any of the three models. CONCLUSION: In a randomized, controlled laboratory study, the quaternary lidocaine derivative, QX-314, concentration-dependently and reversibly produced long-lasting local anesthesia with a slow onset in animal models in vivo. The authors' results raise the possibility that quaternary ammonium compounds may produce clinically useful local anesthesia of long duration in humans and challenge the conventional notion that these agents are ineffective when applied extracellularly.